CN110168093B - Kit for transfecting intracellular parasites and application thereof - Google Patents
Kit for transfecting intracellular parasites and application thereof Download PDFInfo
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- CN110168093B CN110168093B CN201780001027.4A CN201780001027A CN110168093B CN 110168093 B CN110168093 B CN 110168093B CN 201780001027 A CN201780001027 A CN 201780001027A CN 110168093 B CN110168093 B CN 110168093B
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Abstract
The invention relates to a kit for transfecting intracellular parasites and application thereof, wherein the kit comprises polyethyleneimine, and further comprises saponin and/or polyethylene glycol octyl phenyl ether. The kit provided by the invention has the advantages of quick transfection, high transfection efficiency and high transfection power; the time consumption for obtaining the positive insect strain after transfection is short, and long-time drug screening is not needed; only a very small amount of chemical transfection reagent is needed, and expensive transfection instruments are not needed; the cost of raw materials is low, and the cost of the single-transfection reagent is low; has the potential of transfecting a plurality of gene expression vectors at one time.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a kit for transfecting an intracellular parasite and application thereof, and specifically relates to a kit for transfecting the intracellular parasite and a method for transfecting the intracellular parasite.
Background
AIDS, tuberculosis and malaria are the three most deadly infectious diseases in the world. According to World Health organization statistics, 43.8 tens of thousands of people die from Malaria by 2015, with 90% of the mortality concentrated in africa, 7% occurring in southeast asia and 2% occurring in eastern Mediterranean (World Health organization.world Malaria Report 2015). Clinical malaria occurs mainly due to protozoan parasitic infection, which is a human plasmodium falciparum (Plasmodium falciparum, p.falciparum) in the erythrocyte stage. To date, there are many unknown areas of basic biological research on p.falciparum, limiting the development of antimalarial drug therapies and malaria vaccines.
In fact, the function of about 50% of the plasmodium falciparum genes is still unknown (Webster WA, mcFadden GI. From the genome to the phenome: tools to understand the basic biology of Plasmodium falciparum. J Eukaryot Microbiol.2014,61 (6): 655-71.). Various genetic tools have been used for the study of gene function in red-phase plasmodium falciparum, but have been slow in progress (de Koning-Ward TF, gilson PR, crabb bs. Advances in molecular genetic systems in, nat Rev microbiol 2015,13 (6): 373-87.Birnbaum J,Flemming S,Reichard N,Soares AB,Mes en-rami rez P, jonscher E, bergmann B, spielmann T.A genetic system to study Plasmodium falciparum protein function. Nat methods 2017,14 (4): 450-456.). The unsuccessful genetic manipulation of specific genes in plasmodium bodies is often caused by the fact that the transfection efficiency of the plasmodium is low and the survival rate of the plasmodium after transfection is low. Although researchers developed various alternative methods for transfection of malaria parasites, spontaneous absorption of DNA based on erythrocyte electroporation was still the preferred way to introduce exogenous DNA into P.falciparum in the erythrophase (Deitsch K, driskill C, wellems T. Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes. Nucleic Acids Res.2001,29 (3): 850-3.Skinner-Adams TS, lawrei PM, hawthorne PL, gardiner DL, trenholme KR. Comprison of Plasmodium falciparum transfection methods. Malar J.2003, 2:19.). However, this electroporation transfection method requires long-term culture and multiple rounds of drug screening to obtain positive transgenic insect strains (Birnbaum J, flemming S, reichard N, soares AB, mes en-RamI rez P, jonscher E, bergmann B, spielmann T.A genetic system to study Plasmodium falciparum protein function. Nat methods.2017,14 (4): 450-456.).
"Lu et al 2016; birnbaum et al 2017 "reports that simple knock-in of Green Fluorescent Protein (GFP) gene at the nonessential gene locus of the P.falcicut genome also takes several tens of days to obtain positive insect strains (Lu J, tong Y, pan J, yang Y, liu Q, tan X, zhao S, qin L, chen X.A redesigned CRISPR/Cas9system for marker-free genome editing in Plasmodium falcibaum.Parasit vectors.2016,9:198.Birnbaum J,Flemming S,Reichard N,Soares AB,Mes en-RamI rez P, jonscher E, bergmann B, spielmann T.A genetic system to study Plasmodium falciparum protein function. Nat methods.2017,14 (4): 450-456.). Moreover, this method requires expensive electroporation transfection apparatus and matched electrotransfection solution (cytomix) to perform, the amount of DNA required is also large, several hundred micrograms of DNA molecules are consumed for one transfection, red blood cells for DNA loading and plasmodium for transfection are prepared separately, and frequent exchange of solutions is required at the early stage of transfection, which is relatively time consuming, labor consuming and expensive.
Unlike other species, p.falciparum, which is continuously cultured in vitro, must be parasitic in human erythrocytes to survive (Trager W, jensen jb.human malaria parasites in continuous culture. Science 1976,193 (4254): 673-5.), which means that exogenous DNA needs to pass through the human erythrocyte, plasmodium cell membrane and plasmodium nuclear membrane three-layer membrane system to enter the plasmodium nuclei. Furthermore, spontaneous endocytosis of human mature erythrocytes is very inefficient (Colin FC, schrier SL. Spontaneous endocytosis in human neonatal and adult red blood cells: comparison to drug-induced endocytosis and to receptor-mediated endocytosis. Am J Hematol.1991,37 (1): 34-40.), probably due to the dense erythrocyte membrane scaffold increasing the strength of the membrane lipid bilayer (Lux SE 4th.Anatomy of the red cell membrane skeleton:unanswered questions.Blood.2016,127 (2): 187-99.) and thus preventing internalization of the membrane vesicles. This makes it difficult to achieve transfection of plasmodium in the erythrophase by the conventional chemical transfection methods such as cationic liposome method, cationic polymer method, etc. Nevertheless, spontaneous endocytosis (Hoppe HC, van Schalkwyk DA, wiehart UI, meredith SA, egan J, weber BW. Antimalial quinolines and artemisinin inhibit endocytosis in Plasmodium falciparum. Antimicrob Agents chemther. 2004,48 (7): 2370-8.Smythe WA,Joiner KA,Hoppe HC.Actin is required for endocytic trafficking in the malaria parasite Plasmodium falciparum.Cell Microbiol.2008,10 (2): 452-64.) still occurs during erythroid development, and therefore, how to allow the transfected complex containing the gene sequence to permeate the cell membrane of the host is critical for the success of the chemical transfection method.
Polyethyleneimine (PEI) is a stable cationic polymer widely used for mammalian cell transfection (Longo PA, kavran JM, kim MS, leahy DJ. Transient mammalian cell transfection with Polyethylenimine (PEI). Methods enzymes mol.2013,529: 227-40.) and also for gene transfection of Toxoplasma Toxoplasma gondii of protozoan parasites of the same genus as P.falciparum (Salehi N, peng CA. Gene transfection of Toxoplasma gondii using PEI/DNA polypeptides J Microbiol methods.2012,91 (1): 133-7.). PEI can compress DNA into positively charged particles to bind to anions on the cell surface, after which the PEI/DNA complex is endocytosed and released into the cytoplasm (Sonaway ND, szoka FC Jr, verkman AS. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J Biol chem.2003,278 (45): 44826-31.Huth S,Lausier J,Gersting SW,Rudolph C,Plank C,Welsch U,Rosenecker J.Insights into the mechanism of magnetofection using PEI-based magnetofectins for Gene transfer. J Gene Med.2004,6 (8): 923-36.). Coincidentally, another cationic polymer, polyamide-amine dendrimer (polyamidoamine dendrimer), has been reported to be useful in multiple replicates of transfection of erythroid P.falciparum (Mamoun CB, truong R, gluzman I, akopyants NS, OKkman A, goldberg DE.transfer of genes into Plasmodium falciparum by polyamidoamine dendrimers.mol Biochem Parasitol.1999,103 (1): 117-21.), but subsequent studies found that multiple replicates of transfection of P.falciparum and addition of drug screening to obtain stable transfected strains were unsuccessful (Skinner-Adams TS, lawrie PM, hawthormer PL, gardiner DL, trenholme KR.Comparison of Plasmodium falciparum transfection methods J.2003, 2:19.).
Historically, researchers developed a range of methods of transfection of erythroid p.falciparum. Among the most widely used are electroporation transfection methods in which erythrocytes are loaded with DNA, which rely on expensive electroporation transfection apparatus and transfection reagents, the cost of which is as high as several hundred thousand yuan people's coins, the cost of single transfection reagents is several hundred yuan people's coins, and fresh human erythrocytes used for single transfection and P.falciparum (the infection rate of several hundred microliters exceeds 10% of worm blood) in schizont phase need to be prepared separately, the amount of DNA is too large (several tens to several hundred micrograms), one-time electrotransfection needs to take 2 to 3 hours to complete, and at least about thirty days of in vitro culture and drug screening are needed to obtain positive insect strains, and the transfection efficiency and success rate are very low (about one million of transfection rates). There are few researchers trying to use chemical transfection, but the highest transfection efficiency reported is less than 10%, the success rate of the experiment is very low (almost unrepeatable), and the reagents used are expensive.
In addition to malaria parasites, other species of intracellular parasites face chemical transfection challenges. In order to solve the problem of chemical transfection of human plasmodium falciparum, a method can be developed to perforate the plasmodium falciparum-infected human erythrocyte membrane but not damage the parasitic plasmodium therein, and then the plasmodium falciparum-infected human erythrocyte membrane is permeated and plasmodium transfection is achieved by utilizing particles such as polyethyleneimine/DNA complex.
Disclosure of Invention
Aiming at the existing problems, the invention provides a kit for transfecting intracellular parasites and application thereof, and the problem that the transfection efficiency of the parasites is low and the success rate is low can be solved by adopting the kit to transfect the intracellular parasites, so that a system for co-transfecting a plurality of plasmid DNA is realized.
To achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides a kit for transfecting an intracellular parasite, the kit comprising polyethylenimine;
the kit also comprises saponin and/or polyethylene glycol octyl phenyl ether.
In the invention, the efficient transfection of the gene to the intracellular parasites can be realized through the synergistic effect of the Polyethylenimine (PEI) and the saponin and/or the polyethylene glycol octyl phenyl ether (Triton X-100), the whole process only needs about 30min, and the transfection efficiency can reach more than 50 percent, and can reach 100 percent at most.
According to the invention, the kit comprises PEI and saponin.
In the invention, the inventor finds that the use of Triton X-100 and PEI in combination to transfect parasites can generate certain toxicity to the parasites, so that the death rate of the parasites is high, and the use of saponin and PEI in combination has no toxic or side effect, so that the parasites can survive continuously, and the genes can be expressed in the parasites stably.
According to the invention, the parasite is any one or a combination of any two of plasmodium, babesia, human colpoxella-like parasite (reniform parasite), leishmania or trypanosoma, preferably plasmodium, more preferably plasmodium intracellularly.
In a second aspect, the present invention provides a method of transfecting an intracellular parasite using the kit of the first aspect, comprising the steps of:
(1) Treating parasite-infected cells to be transfected with saponin and/or polyethylene glycol octyl phenyl ether in the kit;
(2) And (3) adding a complex of the transfected gene sequence and polyethyleneimine to the cells to be transfected with the parasite infection after the treatment in the step (1) for incubation transfection.
According to the invention, the saponin and/or the polyethylene glycol octyl phenyl ether may be present in a mass volume ratio of 0.001 to 0.05%, respectively, such as 0.001%, 0.002%, 0.003%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.012%, 0.013%, 0.015%, 0.016%, 0.018%, 0.02%, 0.022%, 0.025%, 0.026%, 0.028%, 0.03%, 0.032%, 0.035%, 0.038%, 0.04%, 0.042%, 0.045%, 0.048% or 0.05%, preferably 0.001 to 0.03%, and more preferably 0.001 to 0.01%.
In the present invention, the inventors found that when the saponin concentration is controlled below 0.03% (wt/v, mass/total volume of solution), parasite-infected cells are relatively intact, and few lysis of cells occurs, especially the concentration of saponin is preferably between 0.001% (wt/v) and 0.01% (wt/v), in which the integrity of parasite-infected cells can be maintained to the maximum extent, i.e. the intracellular parasite activity can be maintained, while also being suitable for subsequent PEI transfection.
According to the invention, the gene sequence is any one or a combination of at least two of a plasmid DNA vector, a DNA expression frame sequence or an RNA expression frame sequence.
In the present invention, the transfection may be simultaneous transfection of a plurality of gene expression vectors, including transient transfection, which is transfection of episomally expressed DNA, and stable transfection, which is transfection of a gene editing system capable of genomic integration.
In the present invention, it is possible that the gene sequences are genes capable of being transfected into parasites, and mainly include: a resistance gene, a reporter gene or a functional gene for drug screening positive transfected insect strains, wherein the reporter gene can be, for example, green Fluorescent Protein (GFP) and/or Red Fluorescent Protein (RFP), luciferase, etc.; the functional genes comprise the functional genes of the plasmodium, which are knocked out, or the functional genes which need to be overexpressed are introduced, and the functional genes can be from any parasites or from other species.
According to the present invention, the inventors have found that the molar ratio of amino nitrogen in the polyethyleneimine to the phosphate group in the gene sequence is very important and has a great influence on the transfection efficiency, and that the molar ratio of amino nitrogen in the polyethyleneimine to the phosphate group in the gene sequence in the present invention is 1 to 16000, for example, may be 1,2, 3, 4, 5, 8,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1500, 1800, 2000, 2300, 2400, 2500, 2800, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 110000, 120000, 130000, 140000, 150000 or 160000, preferably 1 to 2500, further preferably 5 to 100.
In a specific example, the specific calculation of the amount of PEI added according to the ratio of the number of moles of amine nitrogen (N) of PEI to the number of moles of phosphate groups (P) of plasmid DNA is as follows: as an example of the free expression plasmid for transient transfection, the phosphate molar concentration (P) =the weight of plasmid DNA (1. Mu.g)/(base pair of double-stranded plasmid DNA (6669 bp). Times.DNA base pair (sodium salt)) is approximately 0.23pmol, calculated as nitrogen-to-phosphorus ratio (N/P) of 30, and 6.9pmol of amine nitrogen (N) of PEI is required, whereas the PEI used in the present invention contains 11% of N-propionylated amine groups, whereas the average molecular weight of PEI is approximately 25,000g/mol, so the amount of PEI required to be added is (6.9 pmol/0.11). Times.25,000 g/mol.1.6. Mu.g, that is, 1.6. Mu.l of PEI working solution (1. Mu.g/. Mu.l) is required to be added.
According to the invention, the molecular weight of the polyethyleneimine is directly related to the amount of subsequent PEI added, the average molecular weight of PEI is about 25000, and the molecular weight of PEI according to the invention is 1000-750000, for example 1000, 1100, 1200, 1300, 1500, 1600, 1800, 2000, 2200, 2300, 2500, 2800, 3000, 3200, 3500, 3800, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 13000, 15000, 18000, 20000, 22000, 25000, 28000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 70000, 80000, 90000, 100000, 130000, 150000, 200000, 250000, 300000, 350000, 400000, 500000, 55000, 600000, 650000, 700000 or 750000, preferably 10000-30000.
According to the present invention, step (1) is preceded by a step of in vitro culturing and synchronizing the parasites, which are known in the art, and can be selected by the person skilled in the art according to the cultured parasites, without any particular limitation.
Preferably, step (2) is followed by a step of culturing.
According to the invention, the culturing step comprises the following steps: the medium containing the transfection reagent was removed and fresh medium was added for cultivation.
Preferably, the temperature of the culture is 30-40deg.C, for example, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, preferably 35-38deg.C.
Preferably, the time of the cultivation is 48h or more, for example, 48h, 49h, 50h, 51h, 52h, 53h, 54h, 55h, 56h, 58h, 60h, 62h, 63h, 65h, 68h, 70h, 72h, 75h, 78h, 80h, 82h, 85h, 88h, 90h, 92h, 95h, 96h, 98h or 100h, preferably 48 to 96h.
Preferably, the cultivation uses a three-gas incubator.
In the invention, positive transfected insect strains can be obtained without drug screening or only through short-time screening due to high transfection efficiency, and the transient transfection does not need to be screened due to the difference of the transient transfection and the stable transfection, and the stable transfection needs 2-3 weeks of drug screening.
In the invention, the drug screening of the stable transfection is to adopt blasticidin S.
According to the invention, the method comprises the following steps:
(1) In vitro culture and synchronization of parasites;
(2) Treating cells infected by parasites to be transfected with saponin and/or polyethylene glycol octyl phenyl ether in the kit, wherein the mass-volume ratio of the saponin and/or the polyethylene glycol octyl phenyl ether is 0.001-0.05% respectively;
(3) Adding a transfected gene sequence and a polyethyleneimine compound to the cells to be transfected with parasite infection after the treatment in the step (2) for incubation and transfection, wherein the molar ratio of amino nitrogen in the polyethyleneimine to phosphate groups in the gene sequence is 1-16000, and the molecular weight of the polyethyleneimine is 1000-750000;
(4) Removing the culture medium containing the transfection reagent, adding fresh culture medium, and culturing at 30-40deg.C in a three-gas incubator for more than 48 hr to obtain stably expressed insect strain.
As a preferred technical solution, the method comprises the following steps:
(1) In vitro culture and synchronization of plasmodium;
(2) Treating plasmodium infected cells to be transfected by using saponin in the kit, wherein the mass-volume ratio of the saponin is 0.001-0.01% respectively;
(3) Adding a transfected gene sequence and a polyethyleneimine compound into the cells to be transfected with plasmodium infection treated in the step (2) for incubation and transfection, wherein the molar ratio of amino nitrogen in the polyethyleneimine to phosphate groups in the gene sequence is 5-100, and the molecular weight of the polyethyleneimine is 10000-30000;
(4) Removing the culture medium containing the transfection reagent, adding fresh culture medium, and culturing at 35-38deg.C in a three-gas incubator for more than 48-96 hr to obtain stably expressed insect strain.
Compared with the prior art, the invention has the following beneficial effects:
(1) The kit can realize efficient transfection of genes to parasites through the synergistic effect of Polyethylenimine (PEI) and saponin and/or polyethylene glycol octyl phenyl ether (Triton X-100), the whole process only needs about 30min, the transfection efficiency can reach more than 50 percent, the highest transfection efficiency can reach 100 percent, the transfected insect plants can be screened to obtain positive insect plants only at most for 2 to 3 weeks without long-time screening;
(2) The transfection kit only needs a small amount of Polyethyleneimine (PEI) and saponin and/or polyethylene glycol octyl phenyl ether (Triton X-100), has low raw material cost, low cost of single-time transfection reagent, and high efficiency, and can transfect a plurality of gene expression vectors at one time;
(3) The method is simple, convenient to operate and convenient to popularize and apply.
Drawings
FIG. 1 is a map of the episomal expression vector used for transient transfection of P.falciparum strains;
FIG. 2 (A) is a CRISPR-Cas9 gene-based editing pPfInt-Pf47 Site clear vector map for stable transfection of P.falciparum strains, and FIG. 2 (B) is a CRISPR-Cas9 gene-based editing pPfInt-GFP & nanoLuc Donor vector map for stable transfection of P.falciparum strains;
FIG. 3 is a basic flow chart of intracellular parasite chemical transfection;
FIG. 4 (A) shows the results of Giemsa staining of P.falciparum strain prior to gelatin enrichment; FIG. 4 (B) shows the results of Giemsa staining of the P.falciparum strain after gelatin enrichment;
FIG. 5 (A) shows the effect of 0.001% by mass/volume of saponin treatment on P.falciparum3D 7-infected erythrocytes, FIG. 5 (B) shows the effect of 0.01% by mass/volume of saponin treatment on P.falciparum3D 7-infected erythrocytes, FIG. 5 (C) shows the effect of 0.03% by mass/volume of saponin treatment on P.falciparum3D 7-infected erythrocytes, FIG. 5 (D) shows the effect of 0.05% by mass/volume of saponin treatment on P.falciparum3D 7-infected erythrocytes, and FIG. 5 (E) shows the effect of 0.1% by mass/volume of saponin treatment on P.falciparum3D 7-infected erythrocytes;
FIG. 6 shows the fluorescence microscopy detection result of P.falciparum3D7 strain transiently transfected by pPfEps-mCherry free expression vector with saponin and PEI, wherein FIG. 6 (A) shows bright field photographing, FIG. 6 (B) shows photographing result of UV channel after Hoechst33258 dye treatment, FIG. 6 (C) shows photographing result of red fluorescence detection channel, and FIG. 6 (D) shows superposition graph of FIG. 6 (A), FIG. 6 (B) and FIG. 6 (C);
fig. 7 is a fluorescent microscope detection result of a CRISPR-Cas9 gene editing expression vector stably transfected p.falciparum3D7 insect strain, wherein fig. 7 (a) is a bright field photograph, fig. 7 (B) is a photograph result of a green fluorescent detection channel, and fig. 7 (C) is a superposition diagram of fig. 7 (a) and fig. 7 (B);
FIG. 8 (A) is a schematic diagram of pPfEps-mCherry plasmid DNA, FIG. 8 (B) is the result of PCR identification of pPfEps-mCherry 3D7 strain transiently transfected with free expression vector, wherein M represents DNA molecular weight standard, S1, S2 is the PCR identification product of PEI transfected strain after saponin treatment of 0.001% (WT/v), S3, S4 is the PCR identification product of PEI transfected strain after saponin treatment of 0.01% (WT/v), WT is the PCR product of wild type P.falciparum3D7 strain as negative control group, and PC is purified pPfs-mCherry plasmid DNA as positive control group;
FIG. 9 (A) is a schematic diagram of double-crossover recombination of pPfInt-GFP & NanoLuc Donor gene Donor plasmid DNA with the Pf47 gene locus on chromosome 13 of the P.falciparum3D7 genome, and FIG. 9 (B) is a PCR identification result of a stably transfected P.falciparum3D7 insect strain with a CRISPR-Cas9 gene editing expression vector, wherein M represents a DNA molecular weight standard, WT is a PCR product of a wild type P.falciparum3D7 insect strain as a negative control group, and S1 is a PCR product of a P.falciparum3D7 stably transfected insect strain;
FIG. 10 shows Western blot identification of P.falciparum3D7 insect strains transiently transfected with pPfEps-mCherry free expression vector, wherein M represents a DNA molecular weight standard, S1, S2 is a cell lysate of P.falciparum3D7 insect strains transfected with PEI after saponin treatment of 0.001% (WT/v), S3, S4 is a cell lysate of P.falciparum3D7 insect strains transfected with PEI after saponin treatment of 0.01% (WT/v), and WT is a cell lysate of wild type P.falciparum3D7 insect strains;
FIG. 11 shows the detection of NanoLuc luciferase activity of P.falciparum3D7 stably transfected insect strains, wherein the ordinate shows the relative fluorescence signal intensity of NanoLuc luciferase activity detection, the abscissa shows the experimental group, S1-S4 are 4 plasmodium strains stably transfected and expressing GFP & NanoLuc Luciferase fusion protein, respectively numbered GFP & NanoLuc, and WT is P.falciparum3D7 wild type insect strain as a negative control group;
FIG. 12 is the effect of Triton X-100 treatment on P.falciparum3D7 infected erythrocytes at mass to volume ratios of 0.001%, 0.01%, 0.025% and 0.1%, respectively;
FIG. 13 shows the fluorescence microscopy results of transient transfection of P.falciparum3D7 strain with pPfEps-mCherry free expression vector using Triton X-100 in combination with PEI, wherein FIG. 13 (A) shows bright field photographs, FIG. 13 (B) shows photographs of UV channels after Hoechst33258 dye treatment, FIG. 13 (C) shows photographs of red fluorescence detection channels, and FIG. 13 (D) shows a superposition of FIGS. 13 (A), 13 (B) and 13 (C).
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below by the specific embodiments in combination with the accompanying drawings, but the invention is not limited to the examples.
Example 1: in vitro culture and synchronization of human plasmodium falciparum strain p.falciparum3D7
In vitro culture and synchronization of Plasmodium falciparum strain P.falciparum3D7 was performed using standardized conditions (Radfar A, M ndez D, moneriz C, linares M, mari n-Garc I a P, puyet A, diez A, bautita JM.Synchronous culture of Plasmodium falciparum at high parasitemia levels.Nat Protoc.2009,4 (12): 1899-915.): comprising the use of a complete medium: 10.4g/L RPMI 1640, 25mM HEPES,0.5% (wt/vol) albuMAX I,1.77mM sodium bicarbonate, 100. Mu.M hypoxanthine, 12.5. Mu.g/ml gentamycin sulfate, pH adjusted to 7.2,1% hematocrit, three-gas incubator at 37 ℃ (1%O) 2 ,3％CO 2 And 96% N 2 ) Is cultured. The medium was changed once daily and the infection rate was counted by Giemsa staining (Giemsa staining) and after the protozoa infection rate reached 10%, plasmodium was synchronized by sorbitol lysis.
Example 2: plasmid vector transfected with falciparum and related gene sequences
To test the feasibility of chemical transfection, two sets of plasmids were used: one is the episomal expression plasmid for transient transfection, pPfEps-mCherry, as shown in fig. 1, whose major elements include: a fusion expression protein frame of red fluorescent protein mCherry controlled by a promoter sequence of a constitutive promoter 5'-CAM (calmodulin, calmolin, CAM, gene ID: PF3D 7-1434200) Gene of P.falciparum and a terminator sequence 3' -PbDT (bifunctional dihydrofolate reductase thymidine synthase, bifunctional dihydrofolate reductase-thymidylate synthase, DHFR-TS, gene ID: PBANKA-0719300) of P.falciparum, a membrane-export transport signal peptide of KAHRP (knob-associated histidine-rich protein) Gene inserted at the N-terminus of mCherry protein, and a Strep tag protein tag linked with a linking peptide composed of serine (S) and alanine (A) fused at the C-terminus of mCherry protein; an expression cassette of a Blasticidin S resistance Gene (Blastidin-S deaminase, BSD, EC number: 3.5.4.23) controlled by the 5 '-terminal promoter of the translation elongation factor of P.falciparum (5' -PfEf1α, elongation factor 1-alpha, gene ID: PF3D 7-1357000) 3 '-terminal non-coding region of the P.falciparum HRPII Gene (3' -HRP2, histidine-rich protein II, HRPII, gene ID: PF3D 7-0831800) for resistance screening of transgenic plasmodium; and an expression cassette for kanamycin resistance gene (kanamycin phosphotransferase, kanamycin resistance gene, kmR) for positive plasmid selection in E.coli (e.g., DH 5. Alpha., XL-10, stbl3 and NEB Stable plasmid DNA clone strains) and for maintenance of stability of the plasmid in E.coli; and the basic backbone of plasmid DNA and the plasmid DNA replication origin (ColE 1 origin) sequence in E.coli, etc.
The other is a plasmid expression system for stable transfection, as shown in FIG. 2 (A) -FIG. 2 (B), comprising two plasmids of pPfInt-Pf47 Site clear of FIG. 2 (A) and pPfInt-GFP & nanoLuc Donor of FIG. 2 (B), which is constructed based on CRISPR-Cas9 gene editing technology, mainly comprising a vector comprising an expression cassette targeting sgRNA of Pf47 Site and an expression cassette of Cas9 gene, the construction process of which is consistent with the construction constitution of pCBS-Pf47 vector (Lu J, tong Y, pan J, yang Y, liu Q, tan X, zhao S, qin L, chen X.A redesigned CRISPR/Cas9system for marker-free genome editing in Plasmodium falciparum.2016 vector, 2016:198); and a recombinant donor plasmid flanked by 5 '-and 3' -terminal homology arm sequences of Pf47 sites in the GFP and NanoLuc fusion protein expression cassette, in agreement with the construction procedure of the pamm-GFP/rucz vector (Lu J, toly, pan J, yang Y, liu Q, tan X, zhao S, qin L, chen X.A redesigned CRISPR/Cas9system for marker-free genome editing in Plasmodium falciparum. Parametri vector.2016, 9:198), the only difference being that the renilla luciferase gene (renilla luciferase) in the pamm-GFP/rucz vector was replaced with a NanoLuc luciferase gene of higher detection sensitivity (Hall MP, ukch J, binkski BF, valley MP, butler BL, wood MG, otto P, mmerman K, virigs G, machlidint, robers MB, benin MB, electronic device, manger, methylene DH, fan, fig. 11, 4,2012, and the only difference being that the renilla luciferase gene (renilla luciferase) in the pamm-GFP/ruki vector was replaced with a NanoLuc luciferase gene of higher detection sensitivity (Hall MP, ukch J, binkski BF, valley MP); 1848-57.), and a GGPSG short peptide DNA sequence is connected with a green fluorescent protein GFPm3 gene to form a novel fusion protein expression frame.
Example 3: p.falciparum transfection experiments
The transfection flow is shown in fig. 3, and the in vitro culture and the gelatin-enriched erythrocyte inner phase p.falciparum strain are subjected to cell permeation treatment, and then the transfection is incubated, specifically as follows:
since the Knob structure-related histidine-rich protein (KAHRP) located on chromosome p.falciparum No. two is critical for the out-membrane transport of human erythrocyte membrane protein 1 (PfEMP 1), its signal peptide can help its fusion protein to be secreted into the erythroid p.falciparum in vitro. Therefore, when the protozoan infection rate reaches 10%, the gelatin enrichment method is firstly adopted to enrich the complete P.falciparum strain of chromosome 2 (Waterkeyn JG, cowman AF, cooke BM.Plasmodium falciparum: gelatin enrichment selects for parasites with full-length chromosome 2.Implications for cytoadhesion assays.Exp Parasitol.2001,97 (2): 115-8.), and the result is shown in fig. 4 (A) -4 (B), therefore, the enriched strain is more in the most trophoblast stage or schizont stage, which is beneficial to the transfection of PEI/DNA complex particles.
The permeation punch experiments were performed on p.falciparum infected erythrocytes (irbs) as follows: first, 5ml of 1% packed insect blood was transferred to a sterile 15ml centrifuge tube, centrifuged at 350 Xg for 5 minutes at room temperature, and approximately 50. Mu.l of iRBCs were collected. 1ml of washing medium preheated at 37℃was added: 10.4g/L RPMI 1640, 25mM HEPES, 100. Mu.M hypoxanthine, 12.5. Mu.g/ml gentamicin sulfate, 350 Xg, and centrifuged at room temperature for 5 min to remove the supernatant. 500. Mu.l of saponin (Sigma-Aldrich, cat. # S4521) solutions of different concentrations ranging from 0.001% (wt/v) to 0.1% (wt/v), respectively, were added, incubated at room temperature for 3 minutes, and after centrifugation at room temperature for 3 minutes, the supernatant was removed and washed once with 1ml of P.falciparum complete medium pre-heated at 37 ℃. The optimal saponin concentration for cell permeabilization was determined by smear and microscopy of the punch-treated irbs, and as shown in fig. 5 (a) -5 (E), a saponin concentration (wt/v, saponin mass/total volume of solution) of greater than 0.03% resulted in lysis of irbs, which was unsuitable for subsequent PEI/DNA complex particle transfection.
The transfection experiment is completed by using the iRBCs with the optimal saponin concentration to permeate, the saponin concentration is controlled below 0.03% (wt/v), the iRBCs are relatively complete, the condition of cracking of the iRBCs is less, particularly, the concentration of the saponin is best between 0.001% (wt/v) and 0.01% (wt/v), in the concentration range, the integrity of the iRBCs can be maintained to the maximum extent, the activity of plasmodium can be maintained, and the method is also suitable for subsequent PEI transfection.
Transfection
First, a 25kD linear Polyethylenimine (PEI, polysciences, cat# 23966) working solution was prepared: dissolving the weighed PEI with sterile deionized water without endotoxin, which is heated to 80 ℃, cooling to room temperature, adjusting the pH value to 7.0 with hydrochloric acid, fixing the volume of the solution to the required concentration of 1 mug/μl with sterile deionized water without endotoxin, filtering and sterilizing with a 0.22 mu m filter membrane, sub-packaging and storing at-20 ℃ for later use.
The ratio of the number of moles of amine nitrogen (N) of PEI to the number of moles of phosphate (P) of plasmid DNA is calculated by calculation, taking the example of the free expression plasmid pPfEps-mCherry for transient transfection, whose phosphate molar concentration (P) =plasmid DNA weight (1. Mu.g)/(base pair (6669 bp) of double-stranded plasmid DNA. Times.DNA base pair (sodium salt)) is approximately 0.23pmol, calculated as 30 nitrogen to phosphorus ratio (N/P), and PEI (Polysciences, cat. # 23966) used in the present invention contains 11% of N-propionylated amine groups, and the average molecular weight of PEI is approximately 25,000g/mol, so that the required addition amount of PEI is (6.9 pmol/0.11) ×25,000g/mol is approximately 1.6. Mu.g, that is 1.6. Mu.l of PEI working solution (1. Mu.g/. Mu.l) is required to be added. The inventors verified the effect of the molar ratio of amino nitrogen in polyethyleneimine to phosphate groups in the gene sequence (N/P) at 1-16000, and found that transfection was achieved when N/P was 1-16000, with the N/P ratio being 5-100 being the most effective.
1. Mu.g of DNA and the corresponding amount of PEI were added in this order to 200. Mu.l of serum-free DMEM minimal medium (C11995500 BT, gibco) at an N/P ratio of 30, and incubated at room temperature for 20min after thoroughly mixing. During this period, irbs that are permeated through a saponin solution of suitable concentration (0.001% -0.05%) are prepared. After gently mixing the PEI/DNA complex with the prepared iRBCs, it was transferred to a T25 flask (Cat. #169900,Thermo Scientific) with 7ml of P.falciparum complete medium pre-warmed at 37℃and added with an appropriate amount of human fresh erythrocytes rinsed with the washing medium to maintain the packed volume of erythrocytes at about 1%. The flask was placed in a three-gas incubator at 37℃for 8 hours, the medium was changed, and fresh complete medium preheated at 37℃was added to continue the culture in the three-gas incubator at 37 ℃.
The transiently transfected insect strain was not screened for any drug during the culture. While stably transfected insect strains were screened by adding 5.0. Mu.g/mL of the blasticidin S (ThermoFisher Scientific, cat. #R21001) to the medium 48 hours after transfection, and changing the solution once every 24 hours until the concentration of blasticidin S was reduced to 2.5. Mu.g/mL after a significant positive insect strain was observed by fluorescence microscopy.
Example 4: transfection of other parasites
Plasmid vectors similar to example 2 were transfected by the method of example 3 in a similar manner to example 1 for in vitro culture and synchronization of babesia, human colpoxella-like parasites (reniform parasites), leishmania and trypanosoma, and the inventors found that the effect of the transfection of other parasites was comparable to that of plasmodium, and the results of the subsequent experiments were described by the inventors using plasmodium transfection and redundant details were not given here.
Example 5: fluorescence microscope real-time detection
To irbs culture of p.falciparum transfected insect strain was added Hoechst33258 (Invitrogen, cat.#h3569) at a ratio of 1:1000 to a final concentration of 10 μg/ml, incubated for 5 min at 37 ℃, and washed twice with PBS buffer (ph 7.4). The washed irbs were then smeared onto a microscope slide with a low fluorescent background for microscopic observation. Our photographing apparatus used was a PLYMPUS IX73 microscope from Olympus corporation and a 100-fold oil lens (UPLFLN 100X (Oil Immersion) objective lens).
Through identification, a chemical transfection method combining saponin and PEI can be adopted to obtain a transiently transfected positive insect strain within 72 hours, the detection result of a fluorescence microscope is shown in fig. 6 (A) -6 (D), and the statistical result of transfection efficiency is shown in table 1;
after 5 rounds of drug screening (5.0. Mu.g/mL of screening with the addition of blasticidin S started two days after transfection, the liquid was changed every day until a positive strain was obtained) within 2-3 weeks, a stably transfected positive strain was obtained, and the results were as shown in FIG. 7 (A) -FIG. 7 (C).
TABLE 1 P.falciparum3D7 in vitro transient transfection efficiency statistics
It can be seen from Table 1 that the transfection power can reach 100% and the transfection efficiency is above 50%, but that the transfection with PEI alone does not find any positive insect strain.
As can be seen from fig. 6 (a) -6 (D), the fusion protein of the reporter gene mCherry was expressed normally, and the red fluorescence of the reporter gene was indeed derived from the plasmodium strain obtained by chemical transfection; as can be seen from FIGS. 7 (A) -7 (C), the reporter gene GFP is expressed normally, and the green fluorescence of the reporter gene is indeed derived from the plasmodium strain obtained by chemical transfection, and the transfection power can reach 100%.
Example 6: PCR identification
The transiently transfected insect erythrocytes were collected by centrifugation 72 hours after transfection and washed twice with PBS buffer (pH 7.4) for further use. And 5 times of saponin solution with the volume concentration of 0.1 percent is added to lyse the erythrozoon erythrocyte. Incubate gently upside down with mixing at room temperature for 2 min. Plasmodium and related cell membrane fragments released after lysis of the stained erythrocytes were collected by centrifugation at 1900 Xg for 5 min at 4 ℃. The supernatant was removed and washed three times with 3 volumes of PBS buffer (pH 7.4) for use. Wild-type p.falciparum and stably transfected p.falciparum were collected by the same method. Directly using plasmodium cells as templates for PCR amplification and identification. The protocol was developed according to the standard protocol of the PCR kit instructions (Takara, cat. #R050Q). The PCR detection targets and primers are shown in Table 2, and the PCR detection results are shown in FIGS. 8 and 9.
TABLE 2 PCR detection targets and primer lists
As can be seen from FIG. 8, the DNA band size of the reporter gene mCherry is 619bp by using the primer pair P1/P2, and the DNA band size of the bsd gene is 399bp by using the primer pair P3/P4; as can be seen from FIG. 9, the PCR product of the wild P.falciparum3D7 strain is used as a negative control group, and the amplified fragment size is 1390bp; s1, P.falciparum3D7 stable transfected insect PCR product with amplified fragment size 4293bp, positive transfected insect strain was obtained both transiently (FIG. 8) and stably (FIG. 9).
Example 7: SDS-PAGE protein gel electrophoresis and Western blot detection
Using the above method for separating plasmodium from irbs, 20 μl of the transfected isolated strain was collected, 100 μl of 2×SDS-PAGE protein loading buffer (Takara, cat. # 9173) was added, and the mixture was resuspended and transferred to a 1.5ml EP tube (Eppendorf tube). Heated at 100℃for 5 minutes. After cooling, centrifuging at 13,000Xg at room temperature for 1 min, and collecting supernatant to obtain the plasmodium total protein SDS-PAGE electrophoresis sample. The appropriate amount of sample was directly taken and analyzed by 10% SDS-PAGE gel electrophoresis or stored at-20℃for later use.
Before Western blot analysis, the prepared plasmodium total protein samples were separated by 10% SDS-PAGE gel electrophoresis, and the protein bands after SDS-PAGE gel electrophoresis were transferred to a PVDF membrane (BioRad, cat.# 1620177) of a suitable size by a wet transfer system (Tanon, china). The transferred PVDF membrane was blocked with 5% BSA (in TBST buffer) for 2 hours at room temperature. Membrane was washed 3 times (6 minutes each) with ice-chilled TBST buffer, either murine primary antibody (Anti-mCherry Anti-body (diluted 1:1000 with TBST buffer, biovision, cat. # 5993) or Anti-GAPDH Anti-body (diluted 1:2000 with TBST buffer, abcam, cat. # ab 125247) was added, incubated for 1 hour at room temperature, horseradish peroxidase (HRP) -labeled goat Anti-mouse IgG H & L secondary antibody was added 3 times (6 minutes each) with ice-chilled TBST buffer, diluted 1:5000 with TBST buffer, abcam, cat. # ab 205719), and incubated for 1 hour at room temperature. The result of Western blot analysis using a chemiluminescent detector (tan, china) at different exposure times is shown in fig. 10, after washing the membrane 3 times (6 minutes each) with ice-chilled TBST buffer, adding ECL reagent (Millipore, cat.#wbkls0500), incubating at room temperature for 5 minutes in the dark.
As can be seen from FIG. 10, excluding the KAHRP signal peptide sequence, the mCherry- 'SA' Linker-Strep tag fusion protein had a predicted average molecular weight of 27.9kDa, while the housekeeping gene GADPH protein had a predicted average molecular weight of 36kDa, which revealed that the expression of the reporter gene was successfully detected in the chemically transfected insect strain.
Example 8: nanoLuc luciferase activity assay of P.falciparum3D7 stable transfected insect strain
A black 96-well plate (ThermoFisher Scientific, cat. # 07-200-565) was used, 100. Mu.l of sterile deionized water was added to the detection wells, 3. Mu.l of red blood cells (infection rate: about 10%) infected with the P.falciparum3D7 stably transfected insect strain were each aspirated, and the mixture was added to the detection wells containing sterile deionized water, followed by thoroughly mixing. The reaction substrate for the NanoLuc luciferase was prepared according to the NanoLuc luciferase assay kit (Promega, cat.#n1110) instructions, i.e., the substrate and enzyme reaction buffer were diluted and mixed uniformly in a 1:50 ratio. 100. Mu.l of the prepared substrate for nanoLuc luciferase was added to each well of the sample containing P.falciparum3D 7-infected erythrocytes, and after 3 minutes of reaction at room temperature, chemiluminescent signals (λmax=460 nm) were read by a microplate reader (BioTek Synergy H1), and the results of the experiment were recorded and counted, as shown in FIG. 11.
As can be seen from FIG. 11, the fusion protein of GFP and nanoLuc luciferase of the P.falciparum3D7 stable transfected insect strain was expressed normally.
Example 9: experiments with Triton X-100 and PEI co-transfection of P.falciparum3D7
In comparison with example 3, the conditions and method were the same as in example 3 except that Triton X-100 was used and the mass/volume ratio of Triton X-100 was 0.001% -0.01%.
First, as in example 3, the well-perforated irbs were smear and microscopic to determine the optimal Triton X-100 concentration for cell permeabilization.
As shown in FIG. 12, triton X-10 concentrations (wt/v, saponin mass/total solution volume) greater than or equal to 0.01% resulted in lysis of iRBCs and was unsuitable for subsequent PEI/DNA complex particle transfection.
The transfection experiment is completed by using irbs with optimal concentration of Triton X-100 to permeate, the concentration of Triton X-10 is controlled below 0.01% (wt/v), the irbs cells are relatively complete, few cells are subject to lysis, and particularly, the concentration of saponin is best between 0.001% (wt/v) and 0.005% (wt/v), in the concentration range, the integrity of irbs can be maintained to the maximum extent, the activity of plasmodium can be maintained, and the method is also suitable for subsequent PEI transfection.
As in example 3, transient transfection with the episomal expression plasmid pPfEps-mCherry, as shown in FIGS. 13 (A) -13 (D), was carried out, although Triton X-100 and PEI were also capable of transfecting P.falciparum3D7 and positive insect strains were observed within 72 hours after transfection, subsequent observations found that the transfection of Triton X-100 and PEI in combination with parasites resulted in a certain toxicity to the parasites, resulting in a high proportion of parasite deaths, and all insect strains treated with Triton X-100 died within about one week after transfection.
Comparative example 1
In comparison with example 3, the conditions and method were the same as in example 3 except that the step of cell permeation using no saponin was used, and only the transfection step was included.
The results demonstrate that the gene sequence cannot be transferred into intracellular plasmodium.
Comparative example 2
In comparison with example 3, the same conditions and procedures as in example 3 were followed except that transfection was performed without PEI and only saponin treatment followed by incubation was employed.
The results demonstrate that the gene sequence cannot be transferred into intracellular plasmodium.
In conclusion, the kit can realize efficient transfection of genes to parasites through the synergistic effect of Polyethylenimine (PEI) and saponin and/or polyethylene glycol octyl phenyl ether (Triton X-100), the whole process only needs about 30min, the transfection efficiency can reach more than 50 percent, the highest transfection efficiency can reach 100 percent, and transfected insect strains can be screened to obtain positive insect strains without long-time screening and only need 2 to 3 weeks at most.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (8)
1. A kit for transfecting an intracellular parasite, wherein the kit comprises polyethylenimine and saponin;
the mass volume ratio of the saponin is 0.001-0.01%; the molecular weight of the polyethyleneimine is 10000-30000;
the parasite is any one or a combination of at least two of plasmodium, babesia, human Colpodella-like parasite, leishmania or trypanosoma.
2. A method of using the kit for transfection of intracellular parasites as claimed in claim 1, wherein said method comprises the steps of:
(1) Treating the parasite-infected cells to be transfected with the saponin in the kit; the parasite comprises any one or a combination of at least two of plasmodium, babesia, human Colpodella-like parasite, leishmania or trypanosoma; the mass volume ratio of the saponin is 0.001-0.01%;
(2) Adding a complex of the transfected gene sequence and polyethyleneimine to the cells to be transfected by the parasites infected after the treatment in the step (1) for incubation and transfection; the molar ratio of amino nitrogen in the polyethyleneimine to phosphate groups in the gene sequence is 5-100, and the molecular weight of the polyethyleneimine is 10000-30000;
removing the culture medium containing the transfection reagent, and adding fresh culture medium for culture, wherein the culture temperature is 30-40 ℃; the culture time is 48 to h.
3. The method of claim 2, wherein the parasite is a plasmodium.
4. The method of claim 2, wherein the gene sequence is any one or a combination of at least two of a plasmid DNA vector, a DNA expression cassette sequence, or an RNA expression cassette sequence.
5. The method of claim 2, further comprising the step of culturing and synchronizing the parasites in vitro prior to step (1).
6. The method according to claim 2, wherein the temperature of the cultivation is 35-38 ℃.
7. The method of claim 2, wherein the time of culturing is 48-96h.
8. The method of claim 2, wherein the culturing is in a three-gas incubator.
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